| Prions are infectious, amyloid, self-propagating protein aggregates that have been identified in evolutionarily divergent members of the Eukarya. It is not yet known whether prokaryotes can support the formation of prion aggregates. In an exploratory study using the Saccharomyces cerevisiae prion protein, Sup35, we found that infectious protein aggregates can indeed form in bacterial cells, as assayed by both biochemical and genetic means. Further, we found that the formation of Sup35 prion aggregates in the bacterial cell is greatly stimulated by the presence of a second prion protein. The inducibility of Sup35 aggregation by a second prion protein is known as the PIN effect and is a requirement for the formation of infectious Sup35 aggregates in yeast cells, though completely dispensable for the formation of infectious Sup35 aggregates in vitro. Thus, the bacterial system recapitulates the in vivo requirements for yeast prion protein conversion despite separation by > 2 billion years of evolution. We have also shown that both endogenous bacterial chaperones and transplanted yeast chaperones are capable of influencing the accumulation of infectious Sup35 aggregates in the bacterial cytoplasm. Most notably, we have detected an effect of the yeast protein disaggregase, Hsp104, in the bacterial system. Extending the principle underlying the phenotypes conferred by well-characterized yeast prions, we have developed a transcription-based assay capable of detecting amyloid aggregates in the bacterial cytoplasm. We have applied the assay to isolate a mutant of the non-prion E. coli amyloid surface protein, CsgB. Initial characterization of the behavior of Sup35 in our newly developed assay showed that it conferred a highly variable phenotype on the bacterial cells that might reflect unstable conversion of Sup35 to the aggregated state in E. coli cells. The work presented in this dissertation demonstrates an E. coli experimental system for the study of known and putative prion proteins. Our findings raise the possibility that endogenous bacterial prion proteins exist and provide a novel in vivo system to facilitate future studies of prion-chaperone interactions. |
